Diversity of Trichinella species in carnivores from Bosnia and Herzegovina

Background In Bosnia and Herzegovina, domestic and wild carnivores represent a significant driver for the transmission and ecology of zoonotic pathogens, especially those of parasitic aetiology. Nevertheless, there is no systematic research of Trichinella species in animals that have been conducted in Bosnia and Herzegovina, even though trichinellosis is considered the most important parasitic zoonosis. The available results of the few studies carried out in Bosnia and Herzegovina are mainly related to the confirmation of parasitic larvae in the musculature of domestic pigs and wild boars or data related to trichinellosis in humans. The objective of our study was to present the findings of a comprehensive investigation into the species composition of Trichinella among 11 carnivorous species within the territory of Bosnia and Herzegovina, as follows: red fox (Vulpes vulpes), grey wolf (Canis lupus), brown bear (Ursus arctos), wildcat (Felis silvestris), pine marten (Martes martes), European badger (Meles meles), weasel (Mustela nivalis), European polecat (Mustela putorius), Eurasian lynx (Lynx lynx), but also dog (Canis lupus familiaris) and cat (Felis catus). Results In the period 2013–2023, carnivore musculature samples (n = 629), each consisting of 10 g of muscle tissue, were taken post-mortem and individually examined using the artificial digestion method. In the positive samples (n = 128), molecular genotyping and identification of parasitic larvae of Trichinella spp. were performed using a PCR-based technique up to the species/genotype level. Positive samples were used for basic PCR detection of the genus Trichinella (rrnS rt-PCR technique) and genotyping (rrnl-EVS rt-PCR technique). The Trichinella infection was documented for the first time in Bosnia and Herzegovina among red foxes, grey wolves, brown bears, dogs, badgers and Eurasian lynx, with a frequency rate of 20.3%. Additionally, the presence of T. britovi infection was newly confirmed in Bosnia and Herzegovina, marking the initial documented cases. Furthermore, both T. britovi and T. pseudospiralis infections were observed in the wildcat population, whereas T. britovi and T. spiralis infections were detected in pine martens. Consistent with previous research, our findings align particularly regarding carnivores, with data from other countries such as Germany, Finland, Romania, Poland and Spain, where T. britovi exhibits a wider distribution (62.5–100%) compared to T. spiralis (0.0–37.5%). T. britovi is more common among sylvatic carnivores (89.0%), while T. spiralis prevails in wild boars (62.0%), domestic swine (82.0%) and rodents (75.0%). Conclusion The results of our study represent the first molecular identification of species of the genus Trichinella in Bosnia and Herzegovina. Additionally, our findings underscore the necessity for targeted epidemiological studies to thoroughly assess trichinellosis prevalence across diverse animal populations. Considering the relatively high frequency of trichinellosis infection in investigated animal species and its public health implications, there is an evident need for establishing an effective trichinellosis surveillance system in Bosnia and Herzegovina.


Background
Despite the undeniable impact of domestic and wild carnivores on the ecology and transmission of zoonotic pathogens and parasites, this area has been very poorly investigated in Bosnia and Herzegovina (B&H).In addition to wild carnivores' diseases of special interest for public health such as rabies, tularemia, brucellosis, echinococcosis, toxoplasmosis and scabies, one should also take into account the public health importance of trichinellosis.Trichinellosis is a globally widespread parasitic disease that is transmitted by eating raw or semi-raw meat infected with Trichinella larvae [1].Parasites from the genus Trichinella have an indirect development and life cycle, and the infected individual represents both the definitive and transitional host for this parasite.Currently, it has been described 10 species and 3 genotypes of the genus Trichinella, further divided into two clades: encapsulated (Trichinella spiralis, T. nativa, T. britovi, T. nelsoni, T. murelli and T. patagoniensis, T. chanchalensis -T6, T8 and T9) and non-encapsulated species (T.pseudospiralis, T. papuae, T. zimbawensis) [2,3].The main reservoirs and definitive/transitional hosts of these parasites are carnivorous and omnivorous mammals, birds and reptiles, distributed on all continents except Antarctica [2,4].Despite the indisputable impact of man on the environment and, consequently changing the ecosystem, this parasite resists and is the most represented in wild, domestic animals and humans in Eastern Europe, Asia and South America [1].
In Europe, the red fox (Vulpes vulpes) and the raccoon dog are considered the primary reservoir hosts for Trichinella [5].Mustelids (such as badgers, martens etc.) and other carnivores (brown bears, lynxes, grey wolves etc.) may also serve as sources of infection [5].In the Balkan region where Trichinella infections are endemic in domestic pigs, T. spiralis and T. britovi are the two most common species detected in both domestic and wild animals in Serbia, Croatia, Hungary, Romania, Bulgaria and North Macedonia [6].In B&H, human and animal trichinellosis represents a significant public health problem and is the most prevalent food-borne parasitic zoonosis.In the last 20 years, there have been recorded more than 2,000 human cases of trichinellosis, even with a fatal outcome [7].Similarly, the reports from Western Balkans indicate a high annual incidence of human trichinellosis.The number of patients per 100,000 inhabitants in Serbia is estimated to be 5.0 cases, 4.1 cases in B&H, 2.9 to 8.5 cases in Romania, 1.7-4.8cases in Croatia, and 2.4 to 2.9 cases in Bulgaria [8].
Systematically organized identification of Trichinella species using PCR-based molecular techniques was not carried out in B&H ever before.The available results of the few studies are mainly related to the findings of parasitic larvae in the musculature of domestic pigs and wildlife (mostly wild boars), or data related to human trichinellosis.The first molecular identification of T. pseudospirals and T. spiralis from isolated parasitic larvae originating from domestic pigs, and T. britovi from isolated parasitic larvae originating from wild boar in samples originating from B&H, was carried out at the Serbian National Reference Laboratory for Trichinellosis, Institute for the Application of Nuclear Energy (INEP) in 2016 [9].
Considering the scarcity of scientific data on the presence of Trichinella species in animal populations in B&H, the public health importance of Trichinella and substantial wildlife biodiversity, this research has been conceptualized at the University of Sarajevo -Veterinary Faculty.This study aimed to determine the Trichinella species and identify the genotypes of the Trichinella species originating from different species of wild and domestic carnivores from the territory of B&H.

Discussion
Ecological diversity provides various conditions for the development and spread of Trichinella and may influence the frequency and distribution of this parasitic infection in wild carnivores.With 53% forest cover in frequency of trichinellosis infection in investigated animal species and its public health implications, there is an evident need for establishing an effective trichinellosis surveillance system in Bosnia and Herzegovina.Keywords Bosnia and Herzegovina, Trichinella species, Carnivores, Zoonotic B&H, crucial habitat for many wildlife species, studying Trichinella presence provides insights into parasite ecology and human health risks, especially in areas with high biological diversity such as B&H.The identification of Trichinella spp. in several carnivore species from all parts of B&H represents the result of paramount importance in understanding the epidemiology of trichinellosis in B&H.The main finding of our study is reflected in the first identification of T. britovi in the populations of wild carnivores such as red foxes, grey wolves, brown bears, badgers, wildcats, pine martens and Eurasian lynx, but also domestic dogs.Further, there is a first identification of T. pseudospiralis in samples originating from wildcats, and T. spiralis in pine martens.The distribution and frequency of Trichinella spp.among the examined animal species were unequal and statistically significant (p < 0.001).Except for the Eurasian lynx, (only one animal positive for T. britovi), Trichinella spp.were most commonly represented in the population of brown bears.This finding was not significantly different in comparison to the frequency of Trichinella spp. in wildcats, grey wolves, pine martens and badgers, but it was significantly different in comparison to red foxes and domestic carnivores (dogs and cats).Despite these observed differences in frequency, it is important to consider the limitation stemming from the smaller sample size for some of the examined species, which could affect the interpretation of the established frequency results.The intensity of the infection, expressed through the number of Trichinella larvae per gram of sample (LPG), is a very important indicator for determining the possibility of infection and clinical manifestations of the disease.The results of  our study are similar to those obtained in the study conducted by Pozio [10], who pointed out that the intensity of infection with T. britovi (average LPG) was higher in comparison with the infection with T. spiralis.The possible explanation for this fact can be found in the characteristics of the Trichinella species and differences in animal habitat.Wild carnivores (the main hosts of T. britovi) are more represented in areas with higher altitudes (colder climate) compared to domestic pigs and wild boars (the main hosts of T. spiralis).T. britovi is more resistant to freezing temperatures, it can survive up to 11 months in the muscle tissue of carnivores at -15ºC, and in the muscle tissue of pigs at -20ºC for up to three weeks, while the T. spiralis larvae at the same temperatures survive for a few days to a few weeks at most [10].This is supported by the data provided by Blaga et al. [11] for Romania.Searching for 40 isolates, T. britovi (n = 26; 65.0%) was more prevalent and was predominantly found in wild animals (24/28; 86.0%).However, research in Serbia revealed that T. spiralis had a prevalence rate of 77.8% among wild carnivores, particularly red foxes and wildcats, while T. britovi exhibited a prevalence rate of 22.2% in the same hosts.The dominance of T. spiralis among wild animals in Serbia implies the transfer of this species from domestic to wild animals, which may be associated with numerous factors (natural features, forested areas, wild animals, agricultural areas, rural population preference for extensive pig breeding, education, public awareness etc.) [12].

Wildcats
Our study results showed that 12 (41.3%) of investigated muscle samples originating from wildcats (Felis silvestris) were found to be infected with Trichinella spp., with an average LPG of 6.4.Of these, T. britovi larvae were identified in 11 samples, while T. pseudospiralis was identified in only one sample.Data on trichinellosis in wildcats in the Balkans are scarce, i.e., available in a few studies only.Thus, Brglez [13] estimated that the prevalence of trichinellosis in wildcats was 4.5%, while Cvetković et al. [14] highlighted the finding of T. spiralis in the population of wildcats in Serbia.The prevalence of Trichinella spp. in wildcats in Romania was 33.3% and 14.0%, respectively [11,15].Before our study, T. pseudospiralis was confirmed in only a sample originating from the domestic pig from Modriča within B&H [9].In our study, we confirmed the presence of T. pseudospiralis and T. britovi in wildcats from the area of Bosanski Petrovac.Additionally, in other locations including Foča, Bihać, Trnovo, Kalinovik and Drvar, only the presence of T. britovi in wildcats was confirmed (Fig. 1).The number of localities where Trichinella spp.positive wildcats were found in comparison with the number of total investigated localities (6/8) was among the highest, but statistically similar to the ratios found in other host species.The representation of Trichinella spp. in the population of wildcats was significantly higher in comparison to the representation of this parasite in the population of cats and red foxes, but statistically insignificant in comparison to the other investigated animal species.

Eurasian lynx
T. britovi was found in one sample originating from Eurasian lynx (Lynx lynx) with an average LPG of 0.61.The finding of T. britovi in the Eurasian lynx is the first finding of such kind in B&H.The positive sample originated from a Eurasian lynx in the Fojnica area (Fig. 2).Similarly, the T. britovi infection of Eurasian lynx have previously been described in other European countries such as Switzerland, Finland, Estonia, Slovakia and Latvia [13,[16][17][18][19][20][21].

Cat (Felis catus)
None of the cat samples tested positive for the presence of Trichinella larvae.The cats originated from various locations within B&H (Fig. 2); however, the small number of samples is limiting in the context of discussing the true frequency of Trichinella infection in cats.

Grey wolves
The relatively high prevalence of T. britovi in grey wolves (38.8%) in B&H partially supports the results of a previous study which reported that T. britovi is the main causative agent of sylvatic trichinellosis in areas of Europe and Asia with a moderate climate, and besides red foxes, martens, jackals and bears, grey wolves seem to be one of the main reservoirs of this parasite [8].For the areas of the Balkans, Brglez [13] estimated the prevalence of Trichinella spp. in grey wolves at 44.0%, while in Croatia T. britovi was identified in all positive grey wolves samples with a previously established prevalence of 27.0% [22].Similarly, all Trichinella spp.positive grey wolves samples (4/4, 100%) in Serbia were also identified as T. britovi [23].The prevalence of grey wolf trichinellosis in Europe ranged between 8.9% in Italy [24], 31.0%[11] and 40.0%[15] in Romania, up to 97.5% in Russia [25].In our study, T. britovi was confirmed in grey wolves from the locations Foča, Drvar, Bosansko Grahovo, Trnovo and Travnik, characterized by forested areas (Fig. 1).Determining T. britovi larvae in grey wolves did not differ significantly compared to localities where other infected animals were found, although the differences in percentages were very large.The highest frequency of infection was observed in grey wolves from the areas of Foča and Trnovo, while the lowest was in Drvar and Bosansko Grahovo.Concerning other species of animals, the frequency of trichinellosis in grey wolves was significantly higher in comparison to the prevalence of trichinellosis in red foxes and cats.Additionally, with grey wolves, an average LPG of 6.9 (range, 0.6-33) was noted.The range of LPG values, from low to high, was observed among grey wolves collected close to each other.Moreover, even among grey wolves from the same territory, there was a notable variability in LPG levels.Regarding other animal species, the higher frequency of trichinellosis in grey wolves has been found in other studies [26], where the reasons mentioned are related to their apex predator status and longer lifespan compared to foxes and cats.Several studies on Trichinella spp.among wild mesocarnivores in Europe have also reported a higher prevalence of Trichinella spp. in grey wolves and other large mesocarnivores (e.g., Eurasian lynx) compared to sympatric mesocarnivores [17,27].

Red foxes
Our finding of T. britovi in red foxes (15.3%) is supported by the results of other studies in Norway 4.8% [28]; Germany 0.08-0.2%[6]; Romania 7.0%, [11] and 21.5% [29]; Switzerland 1.6% [16]; Spain 0.3% [30]; Poland 5.7% [31] and 4.9% [32]; Serbia 6.7% [23]; Portugal 2.1% [33] and North Macedonia 21.5% [14].According to available data, only Cvetkovic et al. [14] in the territory of Macedonia (21.5%) and Imre et al. [34] in Romania (21.5%) have reported a higher prevalence of red fox trichinellosis.The finding of only T. britovi in red foxes is consistent with studies in Belgium, Italy, Portugal, Switzerland and the Netherlands, while in Ireland and in the Swiss Alps area near the border with France, only the presence of T. spiralis has been confirmed [16,23,33].Positive samples of red foxes have been confirmed at multiple locations in B&H; however, the highest frequency of infection was confirmed in the areas of Trnovo, Kladanj, Ilidža and Bosanska Krupa (Fig. 1).The representation of T. britovi in the population of red foxes was significantly lower than in bears, pine martens, wildcats and grey wolves, while no statistical differences were found in comparison to other animal species.This can be associated with the different number of samples from various carnivore species included in our study.
The mean value of the number of Trichinella spp.larvae found in the musculature of red foxes were 8.8 LPG, and the intensity of infection ranged from 0.1 LPG to 64.0 LPG.This result is consistent with previous studies.Thus, the average LPG for the infection in red foxes ranged from 0.12 LPG (0.02 LPG − 0.6 LPG) [35], 2.75 LPG to 4.4 LPG [36] in Poland, 2.4 LPG (0.3 LPG -216 LPG) in Germany [35], 1 LPG to 15 LPG in North Macedonia [14], and 10.5 LPG in Romania [29].

Dogs
The Trichinella spp.infection was confirmed in 14.2% of samples originating from stray dogs in only two of the nine investigated localities in B&H, specifically in the areas of Trnovo and Prača, characterized by forested areas (Fig. 1).The frequency of T. britovi in dogs was significantly lower than in bears and martens, but insignificant in comparison with other investigated animal species.Although the consumption of dog meat has been nominated as one of the common sources of human trichinellosis worldwide [6], this is not the problem in B&H and the consumption of dog meat cannot be considered a source of infection for humans due to cultural dietary habits.However, since stray dogs are primarily fed with human food leftovers, they can serve as sentinel animals.
Similarly to our findings, dog trichinellosis is present in neighbouring countries and Europe.Thus, 23.3% of investigated dog samples were found to be Trichinella spp.positive in Serbia, identifying T. spiralis in all positive samples [23].In Kosovo, Trichinella spp.larvae were found in 16.0% of investigated dog samples, nominating T. britovi as the predominant parasitic species [37].In Greece, T. spiralis was identified in 4.3% of investigated dog samples [38].In Slovakia, Trichinella spp. was found in 12.8% of investigated dogs, and the vast majority of positive cases were distributed in rural areas in regions with the highest number of red foxes.The authors initiated the research due to the trichinellosis epidemic in Slovakia in 1998, associated with the consumption of smoked pork sausages to which trichinellosis dog meat was added [38].

Brown bears
The high prevalence of T. britovi in brown bears (51.7%) discovered in our study is consistent with the results of other studies of trichinellosis in animals in Europe [6,11,17,35,39,40] but also in humans [41].In ex-Yugoslavia, Brglez [13] stated that 16.8% of brown bears were infected with parasites from the genus Trichinella, and a study from Croatia reported 7.1% of infected brown bears [22].Furthermore, among bears, an average LPG of 6.3 (range, 0.8-32) was observed.The highest frequency of T. britovi infection in brown bears was observed in the area of Kladanj, while the lowest was in the area of Foča (Fig. 1).The prevalence of trichinellosis in the population of brown bears was the highest in comparison to populations of all investigated animal species and significantly higher in comparison to the population of red foxes, dogs and cats.On the other hand, it was not significantly higher in comparison with the population of other investigated animal species.
The consumption of bear meat is not common or widespread among citizens in the area of B&H.However, illegal hunting and inadequate control of the trafficking and consumption of brown bear meat can have implications for public health.Furthermore, since the consumption of brown bear carrion can transmit Trichinella to other animals, it has been suggested that brown bears play a role as long-term reservoirs of Trichinella in the natural environment.

Martens and badgers
Trichinella spp.were determined in 33.3% of samples originating from animals from the family Mustelidae.The most infected animal species was the pine marten with 50.0%Trichinella spp.positive samples.T. britovi was identified in 7 samples, and T. spiralis was identified in only one sample.In addition, T. britovi larvae were found in 33.3% of investigated badgers' samples (33.3%).Our study results were similar to the results of other studies which indicated that T. britovi seem to be predominant Trichinella species in sylvatic carnivores such as martens (Martes martes) and badgers (Meles meles) [39,40,42].On the contrary, Oivanen [17] and Pozio and Murell [39] estimated the frequency of T. spiralis in badgers (Meles meles).Pozio [6] and Gottstein et al. [41] pointed out that the clinical cases of human trichinellosis are most likely transmitted due to the consumption of badger and mink meat, which can represent a public health concern in certain countries.The finding of T. britovi in 33.3% of the investigated samples originating from badgers in our study is similar to the results of the study aimed at investigating the presence of Trichinella spp. in badgers in the Balkans, which reported 25.0% of Trichinella spp.positive badgers' samples [13].
In the region of Bosanski Petrovac (Fig. 1), the finding of T. spiralis in a pine marten in our study represents the first record of such kind in the Balkan area.Similarly, the presence of T. britovi and T. nativa in pine martens in Europe is also an extraordinary finding and, to the best of authors' knowledge, it is only recorded in Latvia and Lithuania [43][44][45].The infrequent detection of T. spiralis in martens indicates that these animals likely have a minimal role in the maintenance or transmission of this parasite in the wild, consistent with earlier findings in various European countries [46,47].
The mean larval burden in species from pine marten and badger (4.9 LPG in pine marten and 2.7 LPG in badger) found in the present study is comparable with the larval burden (6.8 LPG) detected in a previous study in Finland [40].While it was notably higher (59.3 LPG) in pine martens tested in Poland [42].The frequency of finding Trichinella spp. between these two species from the family Mustelidae was not statistically significant.

Conclusion
Our research findings revealed that Trichinella, a causative agent of a parasitic disease, is widely distributed among wild mammals in our country.This indicates that the natural ecosystems of B&H are at risk of Trichinella invasion.Despite the relatively small sample size of animals under study, the results demonstrate the extensive presence of this parasite.Consequently, there is a genuine risk of human infection with Trichinella in areas where the animals were captured.Illegal hunting of wild animals such as brown bears and badgers is often the primary cause of human infections, where the meat of poached mammals is not subjected to veterinary and sanitary examination.This study is of utmost importance, as data on Trichinella prevalence in wildlife is very scarce in our region.In addition to the main finding, our study warrants the need for future specifically designed, epidemiologic studies on trichinellosis, which should investigate trichinellosis in areas and host species that were possibly underrepresented in the present study.Considering the relatively high prevalence of trichinellosis in investigated animal species and its public health implications, there is an evident need for establishing an effective trichinellosis surveillance system in B&H.

Study area
B&H is located in southeastern Europe, in the western part of the Balkan Peninsula.It is a mountainous country within the dominant mountain system of the Dinarides (Dinar Alps) on an area of 51,209.2km 2 , with three basic climate zones: Mediterranean climate near the Adriatic Sea, continental mountainous in the central part of B&H and moderate continental in the northern and eastern parts.Forests and forest land cover 53.0% of the state territory.The percentage of forest cover has decreased by approximately 11.0-12.0% in the last 30 years.B&H is one of the countries in Europe with the greatest biodiversity of plant and animal species [48].

Animal populations and sampling methods
In the period January 2015 -February 2023, the frequency of Trichinella spp.infection was investigated in 11 carnivore species (n = 629), as follows: the red fox (Vulpes vulpes), grey wolves (Canis lupus), brown bear (Ursus arctos), wildcat (Felis silvestris), pine marten (Martes martes), European badger (Meles meles), weasel (Mustela nivalis), European polecat (Mustela putorius), Eurasian lynx (Lynx lynx), but also a dog (Canis lupus familiaris) and a cat (Felis catus).The sampling procedure was conducted following the current legal norm [49], and in cooperation with veterinary and hunting organizations.All investigated carnivorous animal species were found deceased by hunting and veterinary organizations, except for red foxes, which were shot during an official survey on the effectiveness of rabies vaccination.None of these animals were killed for the purposes of our research.Additionally, samples were collected during necropsies of wild and domestic carnivores conducted at the University of Sarajevo -Veterinary Faculty to determine the cause of death, excluding samples from foxes.Muscle samples were collected from carcasses of the adult animals found in 67 areas (municipalities) across the country (Fig. 2).Musculature samples were taken from predilection sites (diaphragm root, tongue, masticatory muscles and tibial muscles).Samples were adequately labelled and kept at temperatures up to 4ºC until examination (no later than two days after admission).All samples were analyzed at the laboratory for parasitology of the University of Sarajevo -Veterinary faculty, accredited by BAS EN ISO/IEC 17025:2018.

Detection and identification ofTrichinellaspp. larvae Digestion method
All collected samples (n = 629) were tested for the presence of Trichinella spp.larvae, i.e., 10 g of musculature, with no connective and fatty tissue, were weighed and examined.All the collected muscle samples were individually examined by artificial digestion method according to the Commission Regulation (EC) no.1375/2015 [50].
Larvae obtained by digestion of each sample were counted under a stereomicroscope (OLYMPUS, model CH20BIMF200) with a sub-stage transmitted light source of adjustable intensity, initially set at a magnification of 15 to 20x to ensure optimal examination of the samples.Magnification was then increased to 40-100x for detailed observation.For each sample, the number of larvae per g of sample (LPG) was determined to estimate the larval load.Collected larvae were washed in distilled water, preserved in 96% ethyl alcohol and stored in a freezer at -20 °C until DNA extraction and PCR analysis.

Extraction of nucleic acids
All molecular analyses of Trichinella spp.larvae were performed at the laboratory for molecular-genetic and forensic investigations of the University of Sarajevo -Veterinary faculty (BAS EN ISO/IEC 17025:2018).The DNA extraction was done from both the referent strain larvae as well as the sampled larvae of Trichinella spp.Before the extraction, each larva was homogenized by vortexing with two glass beads for 10 min.The extraction was performed using DNeasy Blood & Tissue Kit® (Qiagen, Hilden, Germany), according to the manufacturer's instructions.

Detection ofTrichinellagenus by real-time qPCR
The detection of a highly conserved mitochondrial smallsubunit ribosomal RNA gene (rrnS) of the Trichinella genus was performed by an SYBR-Green Real-Time qPCR as described in Cuttel et al. [53].Both primers were used at the final concentration of 300 nm and 5 µL of the extracted DNA was used for each reaction.The extracted DNA of a reference strain T. britovi was used as a positive control.The PCR reaction and melting curve analysis were done with Power SYBR® Green RNA-to-CT Kit (Applied Biosystems, USA), according to the manufacturer's instructions, on a Stratagene Mx3005P® thermocycler (Agilent Technologies, USA).The thermoprofile consisted of polymerase activation at 90 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s, one final extension at 72 °C for 10 min and a melting curve analysis in which the temperature was subsequently increased from 60 to 95 °C, with an increment of 0,5 °C/cycle.Fluorescence signals were measured and collected at the end of each extension step, and continuously during melting point analysis.

Genotyping ofTrichinellaspecies by multiplex real-time qPCR
All samples that yielded positive results for Trichinella genus real-time qPCR were tested further to identify the species.Specifically, the multiplex qPCR used was to discriminate between species T. spiralis, T. pseudospiralis, T. britovi and T. nativa in a single run.The primers for discrimination between T. spiralis, T. pseudospiralis and T. britovi were as described in Guenther et al. [51], and for T. nativa were as described in a protocol by European Union Reference Laboratory for Parasites [52].The amplification kit, thermoprofile and primer concentrations were the same as the ones described for the detection of the Trichinella genus.The results were interpreted by analysing the melting point temperatures of amplicons, which were specific for each tested species: 82 °C for T. spiralis, 78.3 °C for T. pseudopiralis, 82.8 °C for T. britovi and 76.5 °C for T. nativa [51][52][53].

Spatial data processing and statistical analysis
The preparation, presentation, manipulation and analysis of spatial data (maps) was performed using GIS software MapInfo Professional version 9.0.Statistical analysis of the results was performed using Minitab 17 software [54].Differences in the frequency of positive samples between animal species or between localities were analyzed using the chi-square test.The test showed a significant effect (p-value < 0.05) in the frequency of positive findings between animal species, and the differences between individual species were further tested by the chi-square test with Yates' correction for continuity.Fisher's exact test was used for comparisons of a small number of cases (n ≤ 50).P-values less than or equal to 0.05 were considered statistically significant.

Fig. 1
Fig. 1 Map of Bosnia and Herzegovina illustrating the locations where positive Trichinella infections were identified.The study map was developed using ArcGIS® software (ESRI, Redlands, CA, USA), version 3.2

Fig. 2
Fig. 2 Map of Bosnia and Herzegovina with Trichinella investigated municipalities (67 in total).The study map was developed using ArcGIS® software (ESRI, Redlands, CA, USA), version 3.2

Table 1
Frequency (%) and intensity of infection (LPG) with Trichinella spp.larvae in animals in B&H

min-max) No. of positive/ investigated localities Felidae
n pos -number of animals infected with larvae of Trichinella spp.; LPG -mean value of the number of larvae of Trichinella spp. in 1 g of musculature (minimum and maximum number of larvae in 1 g); abc -frequency values in the same column that do not contain a common letter mark are significantly different at the p < 0.05 level; n/a -not applicable (when the number of tested samples is n < 5)